Effects of short-term experimental manipulation of captive social environment on uropygial gland microbiome and preen oil volatile composition

IntroductionAvian preen oil, secreted by the uropygial gland, is an important source of volatile compounds that convey information about the sender’s identity and quality, making preen oil useful for the recognition and assessment of potential mates and rivals. Although intrinsic factors such as hormone levels, genetic background, and diet can affect preen oil volatile compound composition, many of these compounds are not the products of the animal’s own metabolic processes, but rather those of odor-producing symbiotic microbes. Social behavior affects the composition of uropygial microbial communities, as physical contact results in microbe sharing. We experimentally manipulated social interactions in captive dark-eyed juncos (Junco hyemalis) to assess the relative influence of social interactions, subspecies, and sex on uropygial gland microbial composition and the resulting preen oil odor profiles.MethodsWe captured 24 birds at Mountain Lake Biological Station in Virginia, USA, including birds from two seasonally sympatric subspecies – one resident, one migratory. We housed them in an outdoor aviary in three phases of social configurations: first in same-sex, same-subspecies flocks, then in male-female pairs, and finally in the original flocks. Using samples taken every four days of the experiment, we characterized their uropygial gland microbiome through 16S rRNA gene sequencing and their preen oil volatile compounds via GC-MS.ResultsWe predicted that if social environment was the primary driver of uropygial gland microbiome composition, and if microbiome composition in turn affected preen oil volatile profiles, then birds housed together would become more similar over time. Our results did not support this hypothesis, instead showing that sex and subspecies were stronger predictors of microbiome composition. We observed changes in volatile compounds after the birds had been housed in pairs, which disappeared after they were moved back into flocks, suggesting that hormonal changes related to breeding condition were the most important factor in these patterns.DiscussionAlthough early life social environment of nestlings and long-term social relationships have been shown to be important in shaping uropygial gland microbial communities, our study suggests that shorter-term changes in social environment do not have a strong effect on uropygial microbiomes and the resulting preen oil volatile compounds

Effect of Levels of Acetate on the Mevalonate Pathway of Borrelia burgdorferi

Borrelia burgdorferi, the agent of Lyme disease, is a spirochetal pathogen with limited metabolic capabilities that survives under highly disparate host-specific conditions. However, the borrelial genome encodes several proteins of the mevalonate pathway (MP) that utilizes acetyl-CoA as a substrate leading to intermediate metabolites critical for biogenesis of peptidoglycan and post-translational modifications of proteins. In this study, we analyzed the MP and contributions of acetate in modulation of adaptive responses in B. burgdorferi. Reverse-transcription PCR revealed that components of the MP are transcribed as individual open reading frames. Immunoblot analysis using monospecific sera confirmed synthesis of members of the MP in B. burgdorferi. The rate-limiting step of the MP is mediated by HMG-CoA reductase (HMGR) via conversion of HMG-CoA to mevalonate. Recombinant borrelial HMGR exhibited a Km value of 132 µM with a Vmax of 1.94 µmol NADPH oxidized minute−1 (mg protein)−1 and was inhibited by statins. Total protein lysates from two different infectious, clonal isolates of B. burgdorferi grown under conditions that mimicked fed-ticks (pH 6.8/37°C) exhibited increased levels of HMGR while other members of the MP were elevated under unfed-tick (pH 7.6/23°C) conditions. Increased extra-cellular acetate gave rise to elevated levels of MP proteins along with RpoS, CsrABb and their respective regulons responsible for mediating vertebrate host-specific adaptation. Both lactone and acid forms of two different statins inhibited growth of B. burgdorferi strain B31, while overexpression of HMGR was able to partially overcome that inhibition. In summary, these studies on MP and contributions of acetate to host-specific adaptation have helped identify potential metabolic targets that can be manipulated to reduce the incidence of Lyme disease

The behavior of RAD51D and XRCC2 in response to drug induced DNA damage and a continuing study of the fly RAD51 paralogs

Repair of DNA damage is one of the most important processes undergone in a dividing cell. This is a two-part study undertaken to better understand some of the proteins involved in the sensing and repair of DNA damage in Drosophila melanogaster. The first portion of this experiment followed two Drosophila Rad51 paralogs, dmRad51D and dmXRRC2, and using constructs tagged with GFP, found that they entered the nucleus in response to drug induced DNA damage. Approximately one hour after the induction of DNA damage via bleomycin, dmRad51D and dmXRCC2 entered the nucleus of the Drosophila culture cells, where they remained for the next three to four hours. Following this period in the nucleus, the cells were visualized moving back into the cytosol. The second portion of this experiment was concerned with the four Drosophila Rad51 paralogs (dmRad51 D, dmXRCC2, Spn B, and Spn D) and two paralogs from Homo sapiens (hsRad51 D and dmRad51 D) and their interactions

The behavior of RAD51D and XRCC2 in response to drug induced DNA damage and a continuing study of the fly RAD51 paralogs

Repair of DNA damage is one of the most important processes undergone in a dividing cell. This is a two-part study undertaken to better understand some of the proteins involved in the sensing and repair of DNA damage in Drosophila melanogaster. The first portion of this experiment followed two Drosophila Rad51 paralogs, dmRad51D and dmXRRC2, and using constructs tagged with GFP, found that they entered the nucleus in response to drug induced DNA damage. Approximately one hour after the induction of DNA damage via bleomycin, dmRad51D and dmXRCC2 entered the nucleus of the Drosophila culture cells, where they remained for the next three to four hours. Following this period in the nucleus, the cells were visualized moving back into the cytosol. The second portion of this experiment was concerned with the four Drosophila Rad51 paralogs (dmRad51 D, dmXRCC2, Spn B, and Spn D) and two paralogs from Homo sapiens (hsRad51 D and dmRad51 D) and their interactions

MrkD1Pfrom Klebsiella pneumoniae Strain IA565 Allows for Coexistence with Pseudomonas aeruginosa and Protection from Protease-Mediated Biofilm Detachment

exterior, single segmental windo

Pseudomonas aeruginosa gshA Mutant Is Defective in Biofilm Formation, Swarming, and Pyocyanin Production

ABSTRACT Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause severe opportunistic infections. The principal redox buffer employed by this organism is glutathione (GSH). To assess the role of GSH in the virulence of P. aeruginosa, a number of analyses were performed using a mutant strain deficient in gshA, which does not produce GSH. The mutant strain exhibited a growth delay in minimal medium compared to the wild-type strain. Furthermore, the gshA mutant was defective in biofilm and persister cell formation and in swimming and swarming motility and produced reduced levels of pyocyanin, a key virulence factor. Finally, the gshA mutant strain demonstrated increased sensitivity to methyl viologen (a redox cycling agent) as well as the thiol-reactive antibiotics fosfomycin and rifampin. Taken together, these data suggest a key role for GSH in the virulence of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous bacterium that can cause severe opportunistic infections, including many hospital-acquired infections. It is also a major cause of infections in patients with cystic fibrosis. P. aeruginosa is intrinsically resistant to a number of drugs and is capable of forming biofilms that are difficult to eradicate with antibiotics. The number of drug-resistant strains is also increasing, making treatment of P. aeruginosa infections very difficult. Thus, there is an urgent need to understand how P. aeruginosa causes disease in order to find novel ways to treat infections. We show that the principal redox buffer, glutathione (GSH), is involved in intrinsic resistance to the fosfomycin and rifampin antibiotics. We further demonstrate that GSH plays a role in P. aeruginosa disease and infection, since a mutant lacking GSH has less biofilm formation, is less able to swarm, and produces less pyocyanin, a pigment associated with infection

Oligonucleotides used in this study.

a<p>Restriction enzyme sites are underlined.</p

A model for the role of acetate in modulating the vertebrate host-specific adaptation and the mevalonate pathway in <i>B. burgdorferi</i>.

<p>Intracellular levels of acetate contribute to the activation of the Rrp2-RpoN-RpoS pathway leading to the adaptation of <i>B. burgdorferi</i> to vertebrate host-specific conditions and provide the initiating substrate for the mevalonate pathway. It appears that acetate plays a central role in modulating host-specific adaptation to central metabolic functions critical for survival of <i>B. burgdorferi</i> under different environmental conditions. The rate-limiting step of the mevalonate pathway, HMG-CoA reductase (HMGR), is a potential chemotherapeutic target that can be exploited to reduce the survival of <i>B. burgdorferi</i> and hence a reduction in the incidence of Lyme disease. Sequence analysis of members of the MP in <i>B. burgdorferi</i> indicated significant similarity with ORFs of the MP that have been characterized in <i>L. monocytogenes, P. mevalonii</i> and <i>S. aureus</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone-0038171-t004" target="_blank">Table 4</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Fraser1" target="_blank">[16]</a>. Based on this analysis, <i>B. burgdorferi</i> appears to possess all open reading frames (ORFs <i>bb0683</i> to <i>bb0688</i>) necessary for the production of IPP via the mevalonate pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Fraser1" target="_blank">[16]</a>.</p

Sequence comparison of HMGR homologs.

<p>ClustalX alignment of HMGR from eight different species, representing both Class II (<i>Borrelia burgdorferi, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus</i>) and Class I (Human, Syrian Hamster, <i>Drosophila melanogaster</i>, and <i>Saccharomyces cerevisiae</i>) shows a high degree of similarity with regard to binding sites for HMG-CoA/HMG-CoA reductase inhibitors (ENVIG - boxes) and NAD(P)H (DAMGXN and GTVGG - triangles). Indicated by stars are conserved residues shown to be important for catalysis of HMGR in <i>Pseudomonas mevalonii </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038171#pone.0038171-Friesen1" target="_blank">[29]</a>.</p